Due to the enormous allelic diversity of the HLA-B locus, it has been difficult to design an unambiguous method for molecular typing of alleles at this locus. Here we describe an alternative approach for the automated sequencing of HLA-B alleles in which each allele is sequenced independently. HLA-B alleles are co-amplified during a locus-specific reverse transcription from RNA followed by PCR. After amplification, alleles are separated using denaturing gradient gel electrophoresis which separates DNA fragments based on their sequence composition. Bands are excised and directly sequenced. The sequences are then aligned to a database of published HLA-B allele sequences, and an allele assignment is made. With the physical separation and independent examination of alleles, we avoid the problems associated with interpreting heterozygous data. Using this method, we analyzed cells of various types from different sources. Samples included immortalized cell lines from the International Histocompatibility Workshop and aboriginal peoples, and blood samples from a panel of cells which were difficult to type using serological techniques. In all cases, an unambiguous typing was assigned to each sample. Key Words MHC, HLA-B, Tissue typing